Several new tolerance induction protocols have brought us closer to achieving allograft survival in the absence of chronic immunosuppression. However, virus infection poses a threat to the allotolerant state because it can disrupt immunoregulation and induce cross-reactive allospecific T cells through a process termed "heterologous immunity." Studies performed by this Program have defined the temporal relationship of viral infection to the success of tolerance induction and to the durability of healed-in grafts. They have revealed the crucial role of alloreactive CD8+ cell deletion and the ability of viral infection to abort this critical process, and have revealed the key role of heterologous immunity after infection as a generator of primed alloreactive memory cells. The theme of this Program is to understand the reciprocal interaction of transplantation tolerance and virus infection. The Program's goal is to understand the mechanisms behind each of the discoveries that we have made using viruses and TLR agonists as probes into the nature of transplantation tolerance. Project 1 will focus on mechanisms by which infection compromises the induction of transplantation tolerance. Project 2 will focus on mechanisms that compromise the maintenance of transplantation tolerance. Project 3 will carry the studies of Projects 1 and 2 to the molecular level. The specific focus of this project is to define the molecular mechanism of CD8 T cell apoptosis. Project 4 will bridge between murine studies of Projects 1-3 and human transplantation immunology. The project will study common human viruses known to affect human allograft survival using "humanized mice." Addition of two new investigators to this revised Program now brings innovative siRNA in vivo delivery technology for blockade of CD40-CD154 interaction in both mice and in human immune systems in humanized mice. The Program will be supported by a Virology and Technology Core that will provide viruses, virological and immunological analyses and new technologies to identify and quantify alloreactive T cells and by a Mouse and Transplantation Core that will provide necessary mouse resources, perform and train Program personnel in transplantation, and monitor infected animals and grafts in biocontainment. The goal of these studies is to discover mechanisms that determine graft survival in the face of infection and innate immune activation. RELEVANCE: This research will investigate mechanisms underlying the induction of transplantation tolerance and graft survival in the absence of immunosuppression and how infection may modulate this process PROJECT 1: Title: - Mechanisms of Transplantation Tolerance Induction Project Leader: GREINER, D PROJECT 1 DESCRIPTION (provided by applicant): Mechanisms underlying the complex interrelationships of infection and graft survival during induction and maintenance of transplantation tolerance are not well understood. Our goal is to understand how infection blocks the induction of peripheral and central tolerance. We have developed several innovative technologies for identifying virus-immune T cells and determining their anti-viral and cross-reactive alloreactivity at the single cell level. We have also developed methods for 1) quantifying alloreactive T cells using a 'synchimera'model based on CD8+ TCR Tg mice, 2) identifying naive and effector alloreactive T cells by their rapid production of cytokines following alloantigen stimulation, and 3) quantifying in vivo CD8 T cell effector function using an in vivo cytotoxicity assay. We will use these techniques with an exciting new technology for in vivo delivery of siRNA to block of CD40-CD154 interaction. These new technologies will allow us to test our overall hypothesis that induction of pro-inflammatory cytokines and IFN1 is a fundamental mechanism by which innate immune activation modulates the induction of peripheral and central tolerance. Specific Aim 1 is to determine mechanisms by which TLR ligation or virus infection modulates the induction of peripheral tolerance. We will test the hypothesis that innate immune activation by TLR agonists or virus infection abrogates the induction of peripheral tolerance through the production of pro-inflammatory cytokines and IFN1. Specific Aim 2 is to determine mechanisms by which TLR ligation or virus infection modulates establishment of hematopoietic chimerism and central tolerance. We will test the hypothesis that the induction of peripheral and central tolerance involves multiple different but overlapping mechanisms. This project should reveal the mechanism(s) by which infection compromises the induction of peripheral and central transplantation tolerance. This Project will interact closely with Project 2 studying the maintenance of tolerance, and Project 3 studying how alloreactive CD8 T cells die by apoptosis following costimulation blockade. These discoveries will be translated to human immune systems in Project 4 using both the Viral and Technology Core and Animal Core as critical resources for the accomplishment of our research goals. RELEVANCE: As new drugs for prolonging graft survival in patients are implemented in the clinic, the safety and efficacy of these new drugs in the face of environmental insults such as infections are not known. This work will identify how infection during transplantation may affect the host immune system and ultimate fate of the graft.